Edited by Joseph M. Jez Reflectin proteins are widely distributed in reflective structures in cephalopods. However, only in loliginid squids are they and the subwavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins' cationic linkers; the resulting charge neutralization overcomes coulombic repulsion to progressively allow condensation, folding, and assembly into multimeric spheres of tunable well-defined size and low polydispersity. Here, we used dynamic light scattering, transmission EM, CD, atomic force microscopy, and fluorimetry to analyze the structural transitions of reflectins A1 and A2. We also analyzed the assembly behavior of phosphomimetic, deletion, and other mutants in conjunction with pH titration as an in vitro surrogate of phosphorylation. Our experiments uncovered a previously unsuspected, precisely predictive relationship between the extent of neutralization of a reflectin's net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparisons of mutants revealed that this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein. Imaging of large particles and analysis of sequence composition suggested that assembly may proceed through a dynamically arrested liquid-liquid phase-separated intermediate. Intriguingly, it is this dynamic arrest that enables the observed fine-tuning by charge and the resulting calibration between neuronal trigger and color in the squid. These results offer insights into the basis of reflectinbased biophotonics, opening paths for the design of new materials with tunable properties. Cephalopods such as squid and octopuses possess an optically dynamic epithelium, enabling complex camouflage and
Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure and function of proteins in response to neuronal, hormonal and environmental signals. We demonstrate here that the low-voltage electrochemical reduction of histidine residues in reflectin A1, a protein that mediates the neuronal fine-tuning of colour reflected from skin cells for camouflage and communication in squids, acts as an in vitro surrogate for phosphorylation in vivo , driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and a newly designed electrochemical cell integrated with an instrument measuring dynamic light scattering, we demonstrate selective reduction of the imidazolium side chains of histidine in monomers, oligopeptides and this complex protein in solution. The formal reduction potential of imidazolium proves readily distinguishable from those of hydronium and primary amines, allowing unequivocal confirmation of the direct and energetically selective deprotonation of histidine in the protein. The resulting ‘electro-assembly’ provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein structure and function in normal physiology and disease. With its abilities to serve as a surrogate for phosphorylation and other mechanisms of charge neutralization, and to potentially isolate early intermediates in protein assembly, this method may be useful for analysing never-before-seen early intermediates in the phosphorylation-driven assembly of other proteins in normal physiology and disease.
Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin – either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.
In this work, we demonstrate electrochemical triggering and assembly of reflectin, the protein that mediates neuronal fine-tuning of color reflected from skin cells used for camouflage and communication in squids. Electrochemical reduction of histidine residues in the protein acts as an in vitro surrogate for phosphorylation in vivo, driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and in situ dynamic light scattering, we demonstrate selective reduction of the imidazolium sidechains of histidine in monomers, oligopeptides and reflectin in acidic solution. The formal reduction potential of imidazolium proves readily distinguishable from those of hydronium and primary amines, allowing unequivocal confirmation of the direct and energetically-selective deprotonation of histidine in reflectin. The resulting “electro-assembly” methodology provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein structure and function. Figure 1
The intrinsically disordered reflectin proteins fill the reflective Bragg lamellae of iridescent cells in squid. In vivo, phosphorylation of the cationic reflectins leads to protein condensation and hierarchical assembly, driving osmotic dehydration of the lamellae and causing enhancement of intensity and tuning of the color of reflected light. In vitro, purified monomeric reflectin protein can be driven to cyclably and tunably assemble by pH‐neutralization or addition of salt, forming spheres of low polydispersity and reproducible size. Analysis of reflectin assembly by dynamic light scattering, x‐ray scattering, and transmission electron microscopy shows that the calibration between charge‐neutralization and assembly size is enabled by the rapid dynamic arrest of particle growth, as controlled by an electrostatic switch spatially distributed across the reflectin chain. Confocal microscopy of fluorescently labeled micron‐sized reflectin assemblies shows that they exhibit internal dynamics that rapidly slow following assembly, suggesting that assembly occurs through a transient liquid‐liquid phase separation that undergoes gelation to form stable protein‐dense condensates. Electron paramagnetic resonance (EPR) analysis shows the initially disordered reflectin monomers form ordered secondary structure that may be critical in the arrest of growth and stabilization of particles. These results provide new insights into the assembly of these unique intrinsically disordered proteins and the biophotonic systems they form, and suggest pathways for the creation of novel tunable biomaterials. Support or Funding Information This research was supported by the U.S. Department of Energy, U.S. Army Research Office, and Institute for Collaborative Biotechnologies.
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